Plant breeders attempting to respond to the constant demand for new varieties of crops displaying improved agronomic characteristics, have recently benefited from several innovations derived from developments in areas of plant cell or tissue culture. For example, cellular selection procedures analogous to those traditionally employed in microbial strain improvement programs, may be applied to plant cells in culture to recover useful genetic variants, anther culture may be used to decrease time necessary to achieve homozygosity, somatic cell hybridization (e.g., protoplast fusion) techniques may be employed as a means for recombining genomes of sexually incompatible species and specific gene sequences may be introduced into plant cells in culture by recombinant DNA methodologies.
More recently, the potential of tissue culture techniques as a tool for generating variation in plants has been appreciated. The tissue culture cycle generally comprises establishing cells or tissue under defined culture conditions. Explants of cotyledons, hypocotyl, stem, leaf, shoot apex, root, young inflorescences, flower petals, petioles, ovular tissues and embryos have been employed as sources of tissue for establishing the relatively dedifferentiated state in culture. (Evans, D. A., et al., Plant Tissue Culture, Academic Press, pp. 45-113 (1981).) While in the unorganized state the cells are allowed to proliferate, thereby expanding amounts of cellular material originally derived from the explant. Finally, plantlets are regenerated from the expanded cell population. This technique, which essentially represents a form of clonal propagation, results in plants which are expected to be genetically identical to the plant from which the explant was obtained.
Contrary to the prevailing dogma of clonal propagation which asserts that the clones should be genetically identical, variation among the regenerated plantlets was occasionally noted. These were, however, usually dismissed as "artifacts of tissue culture". Incidences of these deviations from the desired characteristics displayed by the plants, which were the source of the explants, have been reported in barley (Deambrogio, E. and Dale, P. J., Cereal Res. Comm., 8:417 (1980)), Chrysanthemum, (Jung-Heiliger, H. and Horn, W., Z. Pflanzezuchtg., 85:185 (1980)), lettuce (Sibi, M., Ann. Amelior. Plantes, 26:523 (1976)), maize (Green, C. E., Hort. Sci., 12:7 (1977), oats (Cummings, D. P., et al., Crop Sci., 16:645 (1976)), onion (Novak, F. J., Z. Pflanzenzuchtg., 84:250 (1980)), Pelargonium (Skirvin, R. M., Euphytica, 27:241 (1978)), pineapple (Wakasa, K., Jap. J. Breeding, 29:13 (1979)), potato (Shepard, J. F., et al., Science, 208:17 (1980)), rape (Wenzel, G., In "The Plant Genome", (D. R. Davies & D. A. Hopwood, Eds.), pg. 185 (1980)), rice (Nishi, T., et al., Nature, 219:508 (1968)), sorghum (Gamborg, O. L., et al., Plant Sci Letts., 10:67 (1977)), sugarcane (Heinz, D. J. and Mee, G.W.P., Crop Sci., 9:346 (1969) and Amer. J. Botany, 58:257 (1971)), and tobacco (Sacristan, M. D. and Melchers, G., Mol. Gen. Genet., 105:317 (1969) and Burk, L. G. and Matzinger, D. F., J. Hered., 67:381 (1976)).
The mere reporting of such variation did not necessarily include the recognition that the variation may provide a useful source of selectable or screenable variants for crop improvement programs. The value of such variation for crop improvement has been profferred (Larkin, P. J. and Scowcroft, W. R., Theor. Appl. Genet., 60:197 (1981) and Sibi, M., et al., U.S. Pat. No. 4,003,156).
In U.S. Pat. No. 4,003,156, Sibi, et al. disclose a process for obtaining variants in plants regenerated from tissue culture by means of manipulating the "epigenetic" environment during tissue culture procedure. The process does not induce changes in the genome, does not result in variants displaying classical Mendelian-type segregation, but results in a "vigor" which may be transmitted either through a pollen parent or a seed parent. By contrast, the process of the subject invention permits the induction and recovery of variants displaying a non-Mendelian mode of inheritance and the process of the copending U.S. patent application Ser. No. 525,106 entitled "Tissue Culture of Lycopersicon spp." Applicants: D. A. Evans and W. R. Sharp, the contents of which are incorporated herein, permits the generation and recovery of variants displaying Mendelian inheritance.
Variability has also been found in plants regenerated from protoplasts (Shepard, J. F., Sci. Amer., 246:154-6 (1982)) and protoplast fusion products, i.e., somatic hybrids. Variability in traits encoded by nuclear genes, e.g., leaf morphology and disease resistance in somatic hybrids were described by Evans, et al. (Theor. Appl. Genet., 62:193-198 (1982)), following protoplast fusion and subsequent proliferation of the hybrid callus a cultural situation is present which is analogous to the regeneration of plantlets from explant tissue as outlined above. However, in addition to induction of genetic changes in the tissue culture environment, interactions between respective parental nuclear and cytoplasmic genomes increase the frequency of variation in regenerated plants.